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Journal of Translational Medicine BioMed Central Open Access Research Molecular Etiology of Hearing Impairment in Inner Mongolia: mutations in SLC26A4 gene and relevant phenotype analysis Pu Dai†1, Yongyi Yuan†1, Deliang Huang†1, Xiuhui Zhu2, Fei Yu1, Dongyang Kang1, Huijun Yuan1, Bailin Wu3, Dongyi Han*1 and Lee- Jun C Wong*4 Address: 1Department of Otolaryngology and Genetic Testing Center for Deafness, Chinese PLA General Hospital, Beijing 100853, PR China, 2Department of Otolaryngology, Chifeng Second Hospital, Chifeng City (Inner Mongolia), PR China, 3Division of Genetics and Metabolism, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA and 4Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA Email: Pu Dai - daipu301@vip.sina.com; Yongyi Yuan - yyymzh@163.com; Deliang Huang - huangdl301@sina.com.cn; Xiuhui Zhu - mzhyyy@gmail.com; Fei Yu - playufei@163.com; Dongyang Kang - kangdongyang33@yahoo.com.cn; Huijun Yuan - yuanhj@301hospital.com.cn; Bailin Wu - bai-lin.wu@childrens.harvard.edu; Dongyi Han* - hdy301@263.net; Lee- Jun C Wong* - ljwong@bcm.edu * Corresponding authors †Equal contributors Published: 30 November 2008 Received: 11 August 2008 Accepted: 30 November 2008 Journal of Translational Medicine 2008, 6:74 doi:10.1186/1479-5876-6-74 This article is available from: http://www.translational-medicine.com/content/6/1/74 © 2008 Dai et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The molecular etiology of hearing impairment in Chinese has not been thoroughly investigated. Study of GJB2 gene revealed that 30.4% of the patients with hearing loss in Inner Mongolia carried GJB2 mutations. The SLC26A4 gene mutations and relevant phenotype are analyzed in this study. Methods: One hundred and thirty-five deaf patients were included. The coding exons of SLC26A4 gene were sequence analyzed in 111 patients, not including 22 patients carrying bi-allelic GJB2 mutations or one patient carrying a known GJB2 dominant mutation as well as one patient with mtDNA 1555A>G mutation. All patients with SLC26A4 mutations or variants were subjected to high resolution temporal bone CT scan and those with confirmed enlarged vestibular aqueduct and/or other inner ear malformation were then given further ultrasound scan of thyroid and thyroid hormone assays. Results: Twenty-six patients (19.26%, 26/135) were found carrying SLC26A4 mutation. Among them, 17 patients with bi-allelic SLC26A4 mutations were all confirmed to have EVA or other inner ear malformation by CT scan. Nine patients were heterozygous for one SLC26A4 mutation, including 3 confirmed to be EVA or EVA and Mondini dysplasia by CT scan. The most common mutation, IVS7-2A>G, accounted for 58.14% (25/43) of all SLC26A4 mutant alleles. The shape and function of thyroid were confirmed to be normal by thyroid ultrasound scan and thyroid hormone assays in 19 of the 20 patients with EVA or other inner ear malformation except one who had cystoid change in the right side of thyroid. No Pendred syndrome was diagnosed. Conclusion: In Inner Mongolia, China, mutations in SLC26A4 gene account for about 12.6% (17/135) of the patients with hearing loss. Together with GJB2 (23/135), SLC26A4 are the two most commonly mutated genes causing deafness in this region. Pendred syndrome is not detected in this deaf population. We established a new strategy that detects SLC26A4 mutations prior to the temporal bone CT scan to find EVA and inner ear malformation patients. This model has a unique advantage in epidemiologic study of large deaf population. Page 1 of 12 (page number not for citation purposes)
Journal of Translational Medicine 2008, 6:74 http://www.translational-medicine.com/content/6/1/74 tively[24,29]. Whereas in China, 97.9% EVA patients in Introduction Every year in China, about 30,000 children, compared to simplex families were detected with either biallelic or 840 in UK and one of every one thousand infants in US, monoallelic mutations, of which 88.4% were carrying are born with congenital hearing impairment[1-3]. Hear- biallelic variants and 9.5% with monoallelic mutation. ing impairment is the most common neurosensory disor- Only 2.1% Chinese EVA patients had no mutant SLC26A4 der in human that has an incidence of approximately 1 in allele detected[27]. In addition, the prevalent mutations 1000 children worldwide[4]. About 50–60% of these in different ethnic groups are very different. Campbell et cases have a genetic cause. The most common molecular al. reported T416P and IVS8+1G>A as the two most fre- defects for nonsyndromic autosomal recessive deafness lie quent mutations in northern European population [22], on Connexin 26, a gap junction protein encoded by the while Blons et al. showed a completely different mutation GJB2[5-12]. More than 150 mutations, polymorphisms spectrum that was extremely heterogeneous[23]. In Japa- and unclassified variants have been described in GJB2 to nese, H723R accounted for 53% of the mutant alleles, and account for about 8–40% of molecular etiology of the in Korean, the H723R and the IVS7-2A>G mutation was patients with nonsyndromic hearing impairment http:// the most prevalent mutation accounting for 45.5% of davinci.crg.es/deafness[3]. However, about 80% of the patients with PS or EVA[19,29]. In China, IVS7-2A>G patients with nonsyndromic hereditary deafness in China mutation was the most common form accounting for do not have mutations in GJB2[13]. 57.63% of the mutant alleles[27]. All of the above studies focused on the EVA or Pendred syndrome patients. Pendred syndrome (PS) is the most common form of syn- dromic deafness that accounts for about 10% of heredi- In order to investigate the ratio of EVA or Pendred syn- tary hearing impairment[14]. It is an autosomal recessive drome in Chinese hearing impairment patients and pro- disorder caused by biallelic mutations in SLC26A4 result- vide effective genetic testing and accurate counseling for ing in hearing loss, enlargement of the vestibular aque- hearing loss patients and families in China, we performed duct (EVA) and iodine organification defect in the thyroid SLC26A4 sequence analysis in hearing impairment gland[15]. EVA is always detected in the ears of patients patients in Chifeng City from Inner Mongolia and then with PS by computed tomography (CT) and magnetic res- made a genotype-phenotype correlation analysis. onance imaging (MRI)[16]. EVA is the most common form of the inner ear malformation associated with Materials and methods prelingual or postlingual sensorineural hearing loss and is Patients and DNA samples an important feature of PS[17,18]. EVA may occur alone A total of 135 deaf students from unrelated families of or in combination with an incomplete partition of the Chifeng Special Education School in Inner Mongolia, apical turn of the cochlea as part of Mondini deformity. PS China, were included in this study. Among them, 73 is differentiated from nonsyndromic hearing loss with patients suffered pre-lingual hearing impairment and 28 EVA by the presence of goiter, which usually develops patients suffered post-lingual hearing impairment. The later at around the time of puberty. Since environmental onset of deafness of 34 patients was unclear. Chifeng City and other genetic factors may modulate the effects of Special Education School is the only deaf mute school in SLC26A4 mutations on the development of goiter, the this area. All students with moderate to profound hearing expression of goiter in PS patients is variable and may loss from Chifeng city and within 500 km diameter of its have incomplete penetrance[19]. SLC26A4 encodes an neighboring area come to this school. This cohort of anion (chloride/iodide) transporter transmembrane pro- patients consists of 85 male and 50 female from 3 to 20 tein, pendrin, which is expressed in the thyroid, kidney, years old with the average age of 13.2 ± 3.6. The patients and cochlea[20,21]. DNA sequence analysis identified include 94 of Han, 31 of Mongolian, 7 of Man, and 3 of more than 100 different mutations in SLC26A4[10,15,22- Hui races. This study was performed according to a proto- 27]. The mutation spectrum varies widely among different col approved by the ethnicity committee of the Chinese ethnic groups[10,15,19,23,26-30]. Park and Pryor PLA General Hospital. Informed consent was obtained observed that patients with PS were always associated from all parents prior to blood sampling. Parents were with two mutant alleles in SLC26A4 consistent with auto- interviewed for age of onset, family history, mother's health somal recessive disorder, whereas patients with nonsyn- condition during pregnancy and patient's clinical history dromic hearing loss and EVA might have one or zero including infection, possible head or brain injury and the mutant allele[15,19]. In Caucasian nonsyndromic EVA usage of aminoglycoside antibiotics. In addition, 50 (race cohort, about one third of the patients had two mutant matched) controls with normal hearing were screened for alleles, one third had one mutant allele and one third had SLC26A4 mutations by DHPLC followed by sequencing zero[19]. In Japanese and Korean EVA patients, the pro- analysis. DNA was extracted from peripheral blood leuko- portion of patients having two identified mutant alleles in cytes using commercially available DNA extraction kit SLC26A4 is much higher, 57% and 81%, respec- (Watson Biotechnologies Inc, Shanghai, China). Page 2 of 12 (page number not for citation purposes)
Journal of Translational Medicine 2008, 6:74 http://www.translational-medicine.com/content/6/1/74 (Patient 17) with compound heterozygote of two unclas- Mutational analysis DNA sequence analysis of GJB2, mitochondrial 12S rRNA sified variants, Y375C and R470H, which are most likely and SLC26A4 were performed by PCR amplification of pathogenic (Table 1). Six patients (19 to 24) carry one the coding exons plus approximated 50–100 bp of the SLC26A4 mutant allele, and two patients (18 and 25) flanking intron regions followed by Big Dye sequencing carry a novel unclassified missense variant, I491T and and analysis using ABI 3100 DNA sequencing machine L597S, respectively, that are likely pathogenic due to their (ABI, Foster City, USA.) and ABI 3100 Analysis Software evolutionary conservation and conserved amino acid v.3.7 NT according to manufacturer's procedures. Patients change. Patient 26 carried V659L, a pathogenic mutation with two GJB2 mutant alleles (22 cases) or one dominant that has also been found in a patient with EVA (Patient mutant allele (one case) or mtDNA 1555 A>G mutation 11). The pathogenicity of V659L is reported by Wang et al. (one case) were not further analyzed for SLC26A4 muta- in Chinese enlarged vestibular aqueduct patients[27]. tions. The exons of SLC26A4 of the remaining 111 Each of patients 27 to 29 is heterozygous for an unclassi- patients were sequenced one by one starting from the fre- fied missense variant. Patients 27 and 28 carrying a single quently mutated exons until 2 mutant alleles were identi- conserved amino acid change, I235V and T67S respec- fied. tively, had normal vestibular aqueducts. These two mis- sense variants are probably benign. The novel IVS12-6insT in Patient 29 does not predict a gain or loss of a spice site CT scan and thyroid examination Twenty-nine of 32 individuals who had mutations or var- when analyzed using programe available on http:// iants in SLC26A4 were subjected to temporal bone com- www.fruitfly.org/seq_tools/splice.html. So it is also con- puterized tomography (CT) scan for the diagnosis of EVA sidered benign. Thus, mutations in SLC26A4 were identi- or inner ear malformation based on the criteria of a diam- fied in 19.26% (26/135) patients with hearing eter of greater than 1.5 mm at the midpoint between the impairment in Inner Mongolia, China, 17 with two common crus and the external aperture[31]. To evaluate mutant alleles and 9 with one mutant allele. for Pendred syndrome, the ultrasound scan of thyroid and the thyroid hormone levels were measured in the patients A total of 7 different pathogenic mutations (IVS7-2A>G, positive for SLC26A4 mutations or variants. These proce- E37X, K77I, S391R, N392Y, T410M, H723R) and 5 most dures were performed at the Second Hospital of Chifeng likely pathogenic novel variants (Y375C, R470H, I491T, City, Inner Mongolia, China. Ten patients with hyperthy- L597S, and H723D) were found. The E37X mutation that roidism but normal hearing were enrolled as positive con- results in a premature stop codon and a truncated protein trol for ultrasound scan of the thyroid and the levels of of less than 5% in length is predicted to be deleterious. thyroid hormone. Since perchlorate discharge testing was The H723D mutation is caused by nucleotide substitu- not a general clinical practice in China, it was not used in tion, c.2167C>G, which is predicted to be deleterious this study. since a milder change at the same amino acid residue, H723R that has been found to be the most common path- ogenic mutation in Japanese. Other missense mutations: Results All patients showed severe to profound bilateral sen- K77I, S391R, N392Y, T410M and H723R have been sorineural hearing impairment on audiograms except reported in patients with hearing loss in other stud- Patient 9 in Table 1 whose right ear pure tone average ies[26,27,29]. (PTA) is 55 dB. The most common mutation in our patient cohort is the aberrant splice site alteration, IVS7-2A>G. Eight patients Correlation of genotype with age of onset of deafness The average age of onset of patients with EVA and/or other were homozygotes, 4 patients were compound heterozy- inner ear malformation is 1.56 ± 1.23. The average age of gotes with another mutant allele, and 5 were heterozy- onset of other patients is 0.97 ± 1.42. There is no signifi- gotes without a second mutant allele. The IVS7-2A>G cant statistic difference between the two groups (P value mutation accounts for 58.14% (25/43, counting only the 0.09, t = 1.71). The average age of onset of patients with definite pathogenic and most likely pathogenic variants) SLC26A4 mutations or variants is 1.27 ± 1.10. The average of all SLC26A4 mutant alleles (Table 1). These results sug- age of onset of patients without SLC26A4 mutations or gest that a significant proportion (26/135 = 19.26%) of variants is 1.03 ± 1.24. There is no significant statistic dif- Chinese hearing impairment has molecular defects in ference between the latter groups (P value 0.46, t = 0.727). SLC26A4. SLC26A4 mutations SLC26A4 mutations in control individuals Sequence analysis of SLC26A4 in these 111 patients with In order to determine carrier frequency in general popula- hearing impairment identified 16 patients (1 to 16) with tion, SLC26A4 exons 2–21 of 50 normal hearing individ- two confirmed pathogenic mutations (Table 1), and one uals were analyzed by DHPLC. One IVS7-2A>G Page 3 of 12 (page number not for citation purposes)
http://www.translational-medicine.com/content/6/1/74 Page 4 of 12 (page number not for citation purposes) Table 1: Phenotype and genotype of SLC26A4 gene related hearing impairment in Inner mongilia Patient Age Genotype Phenotype number Allele 1 Allele 2 CT Age of Diamete PTA (L) PTA (R) Thyroid US scan onset r (mm) (dB) (dB) hormone Of thyroid Nucleotid amino category nucleotid amino acid category e Change acid e change change change aEVA 1 17 IVS7-2 aberrant pathogenic IVS7-2 aberrant pathogenic 0.7 3.28 82. 93 normal normal splicing splicing 2 17 IVS7-2 aberrant pathogenic IVS7-2 aberrant pathogenic EVA 2 3.33 103 106 normal normal splicing splicing 3 9 IVS7-2 aberrant pathogenic IVS7-2 aberrant pathogenic EVA 2.5 2.73 93 95 Total T3 normal splicing splicing slightly elevated 4 16 IVS7-2 aberrant pathogenic IVS7-2 aberrant pathogenic EVA 0 2.73 97 97 normal normal splicing splicing 5 10 IVS7-2 aberrant pathogenic IVS7-2 aberrant pathogenic EVA 1 3.64 76 93 normal normal splicing splicing 6 14 IVS7-2 aberrant pathogenic IVS7-2 aberrant pathogenic EVA 2 2.73 96 83 normal normal splicing splicing 7 10 IVS7-2 aberrant pathogenic IVS7-2 aberrant pathogenic EVA 1 2.0 88 95 normal normal splicing splicing 8 8 IVS7-2 aberrant pathogenic IVS7-2 aberrant pathogenic EVA 2 1.64 101 95 normal normal splicing splicing 9 19 IVS7-2 aberrant pathogenic 230A>T K77I pathogenic EVA 4 2.22 71 55 normal normal splicing Journal of Translational Medicine 2008, 6:74 bT410M 10 16 IVS7-2 aberrant pathogenic 1229C>T pathogenic EVA 3 4.55 78 77 normal normal splicing bV659L 11 14 IVS7-2 aberrant pathogenic 1975G>C pathogenic EVA 3 4.19 95 95 normal normal splicing 12 13 IVS7-2 aberrant pathogenic 2168A>G H723R pathogenic EVA 3.5 4.55 96 85 normal normal splicing 13 13 2168A>G H723R pathogenic 109G>T E37X, pathogenic EVA 0 2.89 90 87 normal Cystoid nonsense change mutation bT410M 14 19 2168A>G H723R pathogenic 1229C>T pathogenic EVA 1.5 2.44 107 102 normal normal 15 17 2168A>G H723R pathogenic 2167C>G H723D Unclassifi EVA 0.25 5.46 85 100 normal normal ed variant bT410M 16 14 1173C>A S391R pathogenic 1229C>T pathogenic EVA 0.1 3.33 95 90 normal normal aNA 17 10 1124A>G Y375C Unclassifi 1409G>A R470H Unclassifi Vestibular 0.1 NA NA NA ed variant ed variant and cochlear malformation 18 19 1472T>C I491T Unclassifi EVA and 0.6 4.44 100 100 NA NA ed variant Mondini
Table 1: Phenotype and genotype of SLC26A4 gene related hearing impairment in Inner mongilia (Continued) http://www.translational-medicine.com/content/6/1/74 Page 5 of 12 (page number not for citation purposes) 19 16 IVS7-2 aberrant pathogenic EVA 2 5.46 93 92 Total T3 normal splicing slightly elevated 20 10 IVS7-2 aberrant pathogenic EVA 2 2.66 76 77 normal normal splicing aND 21 17 IVS7-2 aberrant pathogenic 1905G>A E635E Silent 1 84 107 NA NA splicing variant 22 19 1174A>T N392Y pathogenic ND 0 100 100 NA NA anl 23 16 IVS7-2 aberrant pathogenic 1 110 102 NA NA splicing 24 24 IVS7-2 aberrant pathogenic nl 1.1 100 100 NA NA splicing 25 19 1790T>C L597S Unclassifi nl 1.2 100 100 NA NA ed variant bV659L 26 17 1975G>C pathogenic nl 0 98 100 normal normal 27 15 757A>G I253V Unclassifi nl 1 110 108 NA NA ed variant 28 17 200C>G T67S Unclassifi nl 1.3 95 100 normal normal ed variant 29 13 IVS12-6 Intron Unclassifi nl 1 97 100 NA NA insT insertion ed variant 30 16 225C>G L75L Silent ND 0 110 103 NA NA variant 31 20 678T>C A226A Silent nl 1 105 105 NA NA variant 32 18 1905G>A E635E Silent nl 0.7 110 110 normal normal variant Novel mutations are in bold and italic. nl = normal, EVA = enlarged vestibular aqueduct, ND = not determined, NA = not available, CT = computerized tomography, PTA(L) or (R) = pure tone average(left) or (R), IVS7 = intravening sequence 7 (intron 7), IVS12 = intravening sequence 12 (intron 12), Diameter = Diameter at the midpoint between the common crus and the external aperture. Journal of Translational Medicine 2008, 6:74
Journal of Translational Medicine 2008, 6:74 http://www.translational-medicine.com/content/6/1/74 heterozygote and one silent variant 2217A>G (Q739Q) and 68% respectively for mainland China, Taiwanese, were found. Although this control population is too small Korean and Japanese [8,27,29,32,33]. The mutation to reach the final conclusion, the carrier rate of SLC26A4 detection rate in Caucasian EVA patients is much lower, mutation in northern China is estimated to be about 2%. 53 and 40% respectively in UK and Europe [26,34]. In US Polymorphisms in SLC26A4 gene appear to be rare in gen- population, mutations in SLC26A4 account for about one eral population when compared to GJB2 gene. third of the nonsyndromic EVA patients [15]. Patients with Pendred syndrome, however, had higher mutation detection rate in SLC26A4 gene, 90% in a French study SLC26A4 polymorphisms Three novel silent variants were identified; c.1905C>G [23]. (E635E), c.678T>C (A226A) and c.225C>G (L75L). These silent variants are not detected in the 50 control individu- CT scan als. CT scan revealed EVA and/or other inner ear malforma- tion in 20 patients. Sixteen patients (1 to16) had EVA and two pathogenic mutant alleles, consistent with autosomal Comparison of SLC26A4 mutation spectrum in different recessive disorder caused by bi-allelic loss of function of patient population In Asian population, more than 80% of nonsyndromic pendrin protein (Table 1). Patient 17 had common cystic patients with EVA harbored mutations in SLC26A4 cavity of cochlea and vestibule without EVA. She carried [19,27,29,30]. In Taiwan and China, both made up of two novel missense variants Y375C and R470H (Figure >90% Han Chinese, the IVS7-2A>G splice mutation is the 1). Patient 18 had enlarged vestibular aqueduct with most prevalent. In Japan, H723R is the most prevalent. In Mondini dysplasia (Figure 1). He carried a novel I491T Korea, IVS7-2A>G and H723R are the two most prevalent variant. These results suggest that Y375C, R470H and mutations. There seems to be a shift of mutation from I491T are most likely pathogenic. Two patients with one IVS7-2A>G to H723R from China to Japan with Korea in mutant IVS7-2A>G allele had EVA. CT scan results of the middle. Each population has its own rare variants that Patients 21 and 22 (heterozygote IVS7-2A>G and N392Y are not shared (Table 2). Mutations in SLC26A4 is very respectively) were not available (Table 1). The remaining diverse in European and US populations without any patients had normal CT scan. Testing of the 3 most fre- prevalent mutations that account for more than 10% of quent mutations, IVS7-2 A>G, H723R and T410M, can the alleles in patients with Pendred syndrome or EVA lead to finding 80% of patients with EVA or inner ear mal- (Table 2) [15,23,26]. Variants in SLC26A4 gene in Cauca- formation in this cohort sians are rarely overlapped with those found in Asians. Several patients have multiple affected siblings with the same two mutant alleles supporting that EVA is an auto- Frequencies of SLC26A4 mutations in nonsyndromic somal recessive disease. For example, two sisters of patient deafness, EVA, and Pendred syndrome patients CT scan was performed on 29 of the 32 patients listed in 9 with the same genotype (IVS7-2A>G/K77I) and one sis- Table 1. Among them, 20 (69%) had EVA and/or Mondini ter of Patient 6 with homozygous IVS7-2A>G all have dysplasia. Seventeen patients (17/20 = 85%) who har- EVA. The parents of these two families are normal hearing bored two mutations in SLC26A4 gene. had EVA, except individuals and carriers of corresponding SLC26A4 muta- one Patient (patient 17, Y375C and R470H) had vestibu- tions. lar and cochlea malformation. Only 3 out of the 7 patients who carry one heterozygous mutation had EVA, the other Thyroid ultrasound and thyroid hormone assays 4 were normal. All patients who were heterozygous for Thyroid ultrasound was performed to determine presence silent and most likely benign variants were normal on CT or absence of goitre. None of the patients with SLC26A4 scan (Table 1). Since CT scan was performed after geno- mutations or variants was diagnosed goitre. Only one typing, only patients with SLC26A4 mutations or variants patient (Patient 13) with EVA was found cystoid change in received CT scan. 100% of our patients with two mutant the thyroid by ultrasound scan, while there was no change alleles (17/17) and only 33.3%(3/9) of patients with one in the thyroid hormone levels. Thyroid hormone assays mutant allele were confirmed to have EVA manifestation. showed that total T3 was slightly elevated in two patients The frequency of SLC26A4 mutations in our nonsyndro- (Patient 3 and Patient 19), but this abnormity had no mic deafness patients is 19.3% (26/135). Most reported clinical value when evaluated by endocrinologist from studies focused on screening SLC26A4 mutations in the Chinese PLA General Hospital. EVA or Pendred syndrome patients but not in the nonsyn- dromic deafness patients. Discussion Diagnosis of Pendred syndrome EVA requires the evalua- Other Asian studies report high frequency of finding tion of inner ear malformation by temporal bone CT scan. SLC26A4 mutations in patients with EVA, 97.9, 87, 92, Unfortunately, in Chifeng City, Inner Mongolia, China, Page 6 of 12 (page number not for citation purposes)
Journal of Translational Medicine 2008, 6:74 http://www.translational-medicine.com/content/6/1/74 Table 2: SLC26A4 mutation spectrum among different populations aChinese aChinese aTaiwanese aKorean aJapanese aFrench aCaucasian aUS European aTotal 135 NSHI 95 EVA 38 EVA 26 EVA 10 PS + 32 30 PS 100 EVA 31 PS & EVA number of (20 EVA) EVA patients Total mutant 43 (100) 177(100) 57 (100) 45 (100) 57 (100) 50(100) 64 (100) 32 (100) alleles identified % of SLC26A4 15.92 93.16 75 86.5 67.86 83.33 32(64/200) 51.61(32/62) mutation in (43/270) (177/190) (57/76) (45/52) (57/84) (50/60) total IVS7-2A>G 25 (62.5) 102(57.63) 48 (84.2) 9 (20) 2 (3.51) T410M 3 (7.5) 4(2.26) 1 (1.75) 3 (6) 1(1.56) K77I 1 (2.5) 1(0.56) 1 (1.75) H723R 4 (10) 16(9.04) 1 (1.75) 18 (40) 33 (57.9) H723D 1 (2.5) S391R 1 (2.5) 1(1.56) N392Y 1 (2.5) 5(2.82) 1 (1.75) E37X 1 (2.5) 1(0.56) I491T 1 (2.5) Y375C 1 (2.5) R470H 1 (2.5) V659L 2(5) 1(0.56) S448L 1(0.56) 1 (1.75) T721M 1 (1.75) 1 3 (5.3) 1(2) 2(3.13) A372V 2 (3.51) 4 (7) A387V 1(0.56) 2 (3.51) 2111ins5 2 (3.51) 917delT 1 (1.75) 1652insT 1 (1.75) IVS5-1G>A 1 (1.75) IVS8+1G>A 1 (1.75) 2(4) 3(4.69) 2(6.25) 322delC 1 (1.75) S610X 1 (1.75) C565Y 1 (1.75) K369E 1 (1.75) S657N 1 (1.75) S666F 1 (1.75) P123S 1 (1.75) M147V 2(1.13) 3 (6.67) 1 (1.75) IVS9+3A>G 4 (8.89) 365insT 2 (4.44) S28R 1 (2.22) IVS4+4A>G 1 (2.22) P142R 1 (2.22) S166N 1 (2.22) G497S 1 (2.22) IVS14-1G>A 1(0.56) 1 (2.22) IVS15+5G>A 5(2.82) 1 (2.22) E625X 1 (2.22) L676Q 6(3.39) 1 (2.22) Y530H 7(14) 3(4.69) 1(3.13) L445W 5(10) 4(6.25) 2(6.25) IVS14+1G>A 1(0.56) 4(8) G209V 4(8) 1(1.56) 2(6.25) T416P 3(6) L236P 2(6.25) L597S 1 (2.5) 4(12.5) P76L 1(0.56) T94I 3(1.69) P112S 1(0.56) Page 7 of 12 (page number not for citation purposes)
Journal of Translational Medicine 2008, 6:74 http://www.translational-medicine.com/content/6/1/74 Table 2: SLC26A4 mutation spectrum among different populations (Continued) 349delC 1(0.56) 387delC 1(0.56) G197R 1(0.56) G204V 1(0.56) D271G 1(0.56) 916_917insG 2(1.13) G316X 1(0.56) N392S 1(0.56) 1181_1183del 1(0.56) TCT R409H 3(1.69) Q421P 1(0.56) K440X 1(0.56) Q446X 1(0.56) S448X 1(0.56) Q514X 1(0.56) I529S 1(0.56) I532R 2(1.13) N558I 1(0.56) D573Y 1(0.56) 1746delG 1(0.56) R685I 1(0.56) References This study (Wang et al. (Wu et al. (Park et al. (Tsukamoto (Blons et al. (Albert et al. (Pryor et al. 2007) 2005) 2004) et al. 2003) 2004) 2006) 2005) Numbers in the parentheses are the percentages of mutant alleles in total SLC26A4 mutant alleles identified. a All mutations found in Asian populations are listed, Only the mutations that occurred in at least 3 unrelated families of the European and US populations or the mutations that had occurred in other populations are listed to show the diversity of mutations and the lack of prevalent mutations. b total number of chromosome studied = number of patients × 2 the temporal bone CT scan was too expensive to perform based on a) they are located in evolutionarily conserved and there was lack of expertise for temporal bone evalua- regions (Figure 2), b) substituted amino acids are structur- tion. Under these circumstances, SLC26A4 mutation anal- ally and functionally different from amino acids of the ysis may be the only alternative way for the diagnosis of wild type, c) Y375C, R470H, I491T, L597S and H723D EVA, since blood samples can be collected locally and sent have been found in patients with EVA or other forms of elsewhere for DNA analysis. In this study, 100% patients inner ear malformation, and d) they were not present in (17/17) with bi-allelic mutation were confirmed to have our normal controls. EVA by CT scan performed in Chifeng Second Hospital with the help of a specialist from Beijing. Perchlorate dis- It's interesting to note that patient 18 with inner ear mal- charge testing, a routine testing for thyroid function, is not formation carry one missense mutation only, whether the available in most area of China. We use thyroid hormone missense mutation causes dominant negative effect and/ testing and ultrasound scan of thyroid to examine the or specifies a different phenotype is not clear. Three function and structure of thyroid instead. Our results indi- patients (18 to 20) with EVA or other inner ear malforma- cate that none of patients have PS. These may be tion harbored only one mutant allele. It's possible that the explained by a). testing methods were different, b). the second mutant allele has not yet been identified due to a) age of patients undertaking thyroid ultrasound and thy- mutations deep in introns or promoter regions that are roid hormone assays, 3 to 20, average 13.24 ± 3.92, in this not sequenced, b) intragenic exon deletions, c) mutations study may be too young to have symptoms, c). pheno- in genes other than SLC26A4 may involve in the patho- typic diversity due to different genetic background. genesis (digenic). Thus, the mutations in the SLC26A4 gene account for at least 12.6% (17/135) of the patients In this study, we found that SLC26A4 mutations were with nonsyndromic hearing loss, making it as equally detected in nearly 20% of our patients with hearing commonly mutated gene as GJB2 (23/135 no significant impairment with IVS7-2A>G being the most prevalent difference found after statistical analysis, P > 0.05) in mutation. Among the novel variants, Y375C, R470H, patients from Inner Mongolia. I491T, L597S and H723D were considered pathogenic Page 8 of 12 (page number not for citation purposes)
Journal of Translational Medicine 2008, 6:74 http://www.translational-medicine.com/content/6/1/74 may modulate the expression of SLC26A4. Alternatively there may be dominant negative effect. The SLC26A4 mutation spectrum in ChiFeng City, Inner Mongolia is similar to that reported in Chinese popula- tion but different from that of Japanese. There is a gradient shift of the most prevalent mutation from IVS7-2A>G to H723R, respectively, from Chinese to Japanese with both mutations being equally prevalent in Korean. This obser- vation suggests that IVS7-2A>G and H723R mutations may be the ancient mutations in China and Japan respec- tively. The unique rare mutations evolved more recently. A recent study of 100 unrelated patients with EVA in Euro- pean Caucasians by Albert et al. revealed a diverse muta- tion spectrum without prevalent mutations and only 40 patients carried SLC26A4 mutations[26]. Our previous study on the prevalence of GJB2 mutations in Chinese patients with hearing impairment demonstrated that GJB2 mutations were detected in 30.4% of the patients in ChiFeng city. Together, approximately 49.63% (41+26/ 135) of patients with NSHI in ChiFeng city carried muta- tions in GJB2 or SLC26A4 gene. Whereas about 33.1% and 3.5% of European patients with NSHI carried muta- tions in GJB2 and SLC26A4 respectively, with a total of 36.6%, comparable to that in our patient group [35]. It is not clear why the mutations in SLC26A4 account for much lower percentage of patients with EVA in Caucasian patients. Presumably, other genetic factors and environ- mental factors are involved in the pathogenesis of EVA in Caucasians. Figure 1 A 1124A>G/1409G>A. (Patient 17) The striking spot of this study is that a new strategy that A 1124A>G/1409G>A. (Patient 17). The black arrows in the detects SLC26A4 mutations prior to the temporal bone CT picture showed the common cystic cavity of cochlea and CT scan to find EVA patients are established. In China, vestibule. B 1472T>C/wt. (Patient 18). The white arrows in the cost of temporal CT scan is 200 to 300 RMB, because the CT picture showed the hypolastic cochlea (Mondini). The of the relatively high cost, it is not possible to perform black arrows in the CT picture showed EVA. CT scan in every hearing loss patient in molecular epide- miologic study to diagnose EVA. Since 97.9% of Chinese EVA patients carry SLC26A4 mutation [27], SLC26A4 Unlike GJB2 which is a small gene with a lot of missence mutation in hearing loss patients indicates a high possi- variants, SLC26A4 is a relatively large gene with rare mis- bility of EVA. This model presents unique advantage in sense benign polymorphisms or variants. Thus, novel mis- epidemiologic study in large-scale deaf population to sense variant in SLC26A4 is possibly pathogenic. Two find EVA. questions were raised: can the autosomal recessive SLC26A4 mutations cause hearing impairment without Conclusion EVA or other inner ear malformation, and are there other In Inner Mongolia, China, mutations in SLC26A4 gene genes involved in the pathogenesis of hearing loss with account for at least 12.6% (17/135) of the patients with SLC26A4 (digenic). To answer the first question, screen- nonsyndromic hearing loss. Pendred syndrome is not ing of the SLC26A4 mutations in a large NSHI population detected in the Inner Mongolia deaf population. We without EVA is necessary. For the second question, Malin established a new strategy that detects SLC26A4 muta- Hulander reported that the lack of pendrin expression led tions prior to the temporal bone CT scan to find EVA and to deafness and expansion of the endolymphatic compart- inner ear malformation patients. This model has a unique ment in inner ears of Foxi1 null mutant mice [34]. His advantage in epidemiologic study of large deaf popula- observation provides the direct evidence that other genes tion. Page 9 of 12 (page number not for citation purposes)
Journal of Translational Medicine 2008, 6:74 http://www.translational-medicine.com/content/6/1/74 An alignment will displayor nucleotides following symbols denoting the degree of conservation observed in each column: "*" Figure 2 means that the residues by default the in that column are identical in all sequences in the alignment An alignment will display by default the following symbols denoting the degree of conservation observed in each column: "*" means that the residues or nucleotides in that column are identical in all sequences in the alignment. ":" means that conserved substitutions have been observed, "." means that semi-conserved substitutions are observed. The black arrows shows the amino acid related to newly found mutations or variants. Page 10 of 12 (page number not for citation purposes)
Journal of Translational Medicine 2008, 6:74 http://www.translational-medicine.com/content/6/1/74 Competing interests mutations in Japan, including a frequent founder mutation. Hum Genet 2003, 112:329-333. The authors declare that they have no competing interests. 13. Dai Pu, Yu Fei, Han Bing, Yuan Yongyi, Li Qi, Wang Guojian, Liu Xin, He Jia, Huang Deliang, Kang Dongyang, Zhang Xin, Yuan Huijun, Sch- mitt Eric, Han Dongyi, Wong Lee-Jun: The prevalence of the Authors' contributions 235delC GJB2 mutation in a Chinese deaf population. Genet- Pu Dai, Yongyi Yuan and Deliang Huang carried out the ics IN Medicine 2007, 9:283-289. molecular genetic studies, participated in the sequence 14. Reardon W, Coffey R, Phelps PD, Luxon LM, Stephens D, Kendall- Taylor P, Britton KE, Grossman A, Trembath R: Pendred syn- alignment and drafted the manuscript. Xiuhui Zhu carried drome – 100 years of underascertainment? QJM 1997, out temporal CT scan and thyroid hormone assays. 90:443-447. 15. Pryor SP, Madeo AC, Reynolds JC, Sarlis NJ, Arnos KS, Nance WE, Dongyang Kang participated in the sequence alignment. Yang Y, Zalewski CK, Brewer CC, Butman JA, Griffith AJ: SLC26A4/ Fei Yu and Huijun Yuan participated in the design of the PDS genotype-phenotype correlation in hearing loss with study and performed the statistical analysis. Dongyi Han enlargement of the vestibular aqueduct (EVA): evidence that Pendred syndrome and nonsyndromic EVA are distinct and Bailin Wu conceived of the study, and participated in clinical and genetic entities. J Med Genet 2005, 42:159-165. its design and coordination and helped to draft the man- 16. Phelps PD, Coffey RA, Trembath RC, Luxon LM, Grossman AB, Brit- uscript. Lee-Jun Wong reviewed and interpreted the ton KE, Kendall-Taylor P, Graham JM, Cadge BC, Stephens SG, Pem- brey ME, Reardon W: Radiological malformations of the ear in results, drafted and revised the manuscript. All authors Pendred syndrome. Clin Radiol 1998, 53:268-273. read and approved the final manuscript. 17. Valvassori GE, Clemis JD: The large vestibular aqueduct syn- drome. Laryngoscope 1978, 88:723-728. 18. Cremers CW, Admiraal RJ, Huygen PL, Bolder C, Everett LA, Joosten Acknowledgements FB, Green ED, van Camp G, Otten BJ: Progressive hearing loss, This work was supported by Chinese National Nature Science Foundation hypoplasia of the cochlea and widened vestibular aqueducts are very common features in Pendred's syndrome. Int J Pediatr Research Grant (30572015, 30872862), Beijing Nature Science Foundation Otorhinolaryngol 1998, 45:113-123. Research Grant (7062062) to Dr. Pu Dai and Chinese National Nature Sci- 19. Park HJ, Shaukat S, Liu XZ, Hahn SH, Naz S, Ghosh M, Kim HN, Moon ence Foundation Research Grant (30801285) to Dr. Yongyi Yuan. 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